Re: RE: GFP visualization LONG reply

From:

OLaribi@lbl.gov

Hi,
I'm beginning to work with EGFP mice and I've been reading
contradictory stuff regarding the fixation of frozen tissue. Any help?
Thanks. Ouahiba
----- Original Message -----
From: "Montague, Donna C"
Date: Monday, February 24, 2003 10:20 am
Subject: RE: GFP visualization LONG reply
> Ben and interested others:
> Ben asked:
> --"Due to potentially low signal, we believe a biotin-avidin-HRP
> system is necessary for our application, as direct observation
> doesn't seem feasible.We are attempting to analyze rat stromal
> cells expressing GFP, so I wanted to follow-up on any suggestions
> you might have to avoid non-specific binding since the antibody is
> mouse anti-GFP.
> 1.) I also read where you use hydrocarbon solvents. We use
> Xylene here, so I was wondering if you believe this one difference
> could affect our results?
> 2.) Do you perform any antigen retrieval for the Zymed enzyme?
> 3.) Have you observed low background in GFP negative controls?
> 4.) Is this Zymed MAB the best to use, in your opinion? Other
> options?5.) How long do you incubate in primary and at what
> temperature?6.) In what diluent?
> 7.) What blocking buffer might you recommend?"
>
> GFP visualization in paraffin processed tissue can be tricky. The
> following "pearls" are taken from my own experience and reflect
> what we do in our lab. Neither the University of Arkansas for
> Medical Sciences nor I have financial interest in the companies
> whose products are mentioned in this email. That said, this is
> what we do.
>
> Given that we are a full-service research support laboratory and
> receive specimens from different sources over which we have little
> control, we try to ascertain which GFP product (wild-type (wt) or
> variant (e.g. EGFP)) was used in the specimen. If wtGFP was used,
> we process the specimen for paraffin as usual and then attempt to
> visualize the GFP using an antibody. We have used both Zymed's
> antibody and Clontech's. Even though they are recommended for
> western blot, they work well in paraffin. We start with a
> concentration 10 fold less than that recommended for western blot
> (e.g. if the WB recommended concentration is 1:1000, we would
> start with 1:100 and probably bracket with 1:50 and 1:200 for the
> primary).
>
> GFP tags may be found anywhere within the cell, attached to the
> inside or outside of the cell membrane or extracellularly
> depending on the product to which they are attached. Therefore,
> some permeabilization of the cell membrane may be required. Our
> routine procedure for new antibodies is to:
> 1) Fix well in 10 % neutral buffered formalin
> 2) Decalcify with Formic acid or EDTA as appropriate
> 3) Dehydrate in serial alcohols starting with 70% through 100
> % until all fat and water have been removed
> 4) We clear with methyl salicylate for bone and xylene for
> soft tissues.
> 5) We infiltrate with a 50:50 mixture of EM400 (Surgipath) and
> Tissue Prep 2 (Fisher) using three changes
> 6) We embed in 100 % EM400
> 7) Cut at 5 um
> 8) Float onto SuperFrost Plus (for immuno) or UltraStick (for
> IS-PCR).
> 9) Anneal with heat and allow to cool flat.
> 10) Deparaffinize to dH20.
> 11) Immerse in PBS (pH 7.4) containing 0.02 % Triton X-100
> 12) Antigen retrieval (Biogenex Antigen Retrieval Citra 10X
> concentrate, cat # HK086-5K) is accomplished by immersing the
> slides in 80 - 90 oC citrate buffer (pH 6.0) for 15 minutes. We
> never microwave the slides in the buffer but rather heat the
> buffer then place the slides in the hot solution. Recall that
> tertiary and quaternary proteins unfold at temps less than 95oC.
> Steaming or boiling the slides is merely asking for the tissue to
> fall off the slide regardless of the adhesion additive used.
> 13) Slides are rinsed in PBS-Triton
> 14) Block for endogenous peroxidase activity using 3 % H2O2 in
> PBS-Triton or Methanol as you wish. Rinse in PBS-Triton.
> 15) Apply 1 - 5 % host (e.g. if the antibody was made in
> mouse, use mouse serum) serum in PBS-Triton to block non-specific
> binding. (If non-specific staining is still a problem use a
> completely different species, e.g. horse, donkey or goat).
> 16) Apply the primary antibody and incubate at 4 oC overnight
> or at 25 oC for 1 - 3 hours.
> 17) Rinse with PBS-Triton times 2 for 5 minutes each.
> 18) Apply secondary antibody, incubate according to package
> insert usually 20 minutes at room temp is sufficient
> 19) Select and develop chromophore as desired.
>
> For additional info on GFP see the FAQ section of the Histotech
> Home Page at http://www.histology.to/index.html>
> http://www.histology.to/index.html
> Hope this helps.
> Donna Montague
> University of Arkansas for Medical Sciences
> Department of Physiology & Biophysics and
> Center for Orthopaedic Research
> 4301 W. Markham St. # 505
> Little Rock, AR 72205
>
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